A wide variety of methods have been developed for linking molecules together to form conjugates. Of particular interest are biomolecular conjugates that are typically prepared to combine the functionalities of the joined molecules into one construct. One type of biomolecular conjugate combines a biomolecule that specifically binds to another molecule (such as a nucleic acid, an antibody, a lectin or an avidin) and a detectable label (such as a fluorescent label, fluorescent nanoparticle or an enzyme).
Conjugates of antibodies and detectable labels (antibody conjugates) can be used in immunoassays for detecting specific target molecules in biological samples. The antibody portion of such conjugates specifically binds to a target in the sample and the detectable label is utilized to provide a detectable signal that indicates the presence/and or location of the target. One type of conjugate that has become widely used, especially for immunohistochemical analysis, is a conjugate of an antibody and an enzyme (antibody-enzyme conjugate). A detectable signal is generated by adding a substrate to the sample under conditions where the enzyme portion of the antibody-enzyme conjugate converts the substrate to a detectable product (such as a colored, different-colored or fluorescent product) at the site where the antibody portion is bound to its target.
Antibody conjugates are typically prepared using coupling reagents that are characterized by having at least two reactive groups, one of which is reacted with a functional group on the antibody and the other of which is reacted with a functional group on the detectable label. However, coupling can lead to inactivation of either or both of the antibody and the detectable label. In particular, coupling can deactivate antibody-enzyme conjugates through steric effects or because the coupling reagents react with functional groups located on portions of the antibody and/or enzyme that are critical for their specificity and/or catalytic activity. Furthermore, some coupling schemes lead to conjugates that have reduced water solubility.
Coupling schemes that can provide antibody-enzyme conjugates with reduced impairment of antibody specificity and/or enzyme activity are desirable and enable greater sensitivities to be achieved in immunochemical assays such as immunohistochemical assays. Greater sensitivity is of particular importance for automated processes where additional amplification steps are undesirable.